Novel immunotherapy against neuronal and brain tumors

ABSTRACT

The present invention relates to peptides, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated cytotoxic T cell (CTL) peptide epitopes, alone or in combination with other tumor-associated peptides that serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses. The present invention relates to 11 novel peptide sequences and their variants derived from HLA class I and class II molecules of human tumor cells that can be used in vaccine compositions for eliciting anti-tumor immune responses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/865,278, filed Sep. 25, 2015, which is a divisional of U.S. Ser. No.12/180,170, filed Jul. 25, 2008, which claims priority to EP 08005889.4,filed Mar. 27, 2008 and to EP 07014797.0, filed Jul. 27, 2007, whichclaims the benefit of U.S. Provisional Application 60/953,161, filedJul. 31, 2007 and U.S. Provisional Application 61/041,129, filed Mar.31, 2008, the contents of which are incorporated herein by reference intheir entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

A Sequence Listing is submitted herewith as an ASCII compliant text filenamed “2912919-017002_ST25.txt”), created on Nov. 20, 2018, and having asize of 1,981 bytes as permitted under 37 C.F.R. § 1.821(c). Thematerial in the aforementioned file is hereby incorporated by referencein its entirety.

BACKGROUND Field of the Invention

The present invention relates to peptides, nucleic acids and cells foruse in immunotherapeutic methods. In particular, the present inventionrelates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated cytotoxic T cell (CTL) peptideepitopes, alone or in combination with other tumor-associated peptidesthat serve as active pharmaceutical ingredients of vaccine compositionsthat stimulate anti-tumor immune responses. The present inventionrelates to 11 novel peptide sequences and their variants derived fromHLA class I and class II molecules of human tumor cells that can be usedin vaccine compositions for eliciting anti-tumor immune responses.

Description of Related Art

Gliomas are brain tumors originating from glial cells in the nervoussystem. Glial cells, commonly called neuroglia or simply glia, arenon-neuronal cells that provide support and nutrition, maintainhomeostasis, form myelin, and participate in signal transmission in thenervous system. The two most important subgroups of gliomas areastrocytomas and oligodendrogliomas, named according to the normal glialcell type from which they originate (astrocytes or oligodendrocytes,respectively). Belonging to the subgroup of astrocytomas, glioblastomamultiforme (referred to as glioblastoma hereinafter) is the most commonmalignant brain tumor in adults and accounts for approx. 40% of allmalignant brain tumors and approx. 50% of gliomas (CBTRUS, 2006). Itaggressively invades the central nervous system and is ranked at thehighest malignancy level (grade IV) among all gliomas. Although therehas been steady progress in their treatment due to improvements inneuroimaging, microsurgery, diverse treatment options such astemozolomide, and radiation, glioblastomas remain incurable (Macdonald,2001; Burton and Prados, 2000; Prados and Levin, 2000). The lethal rateof this brain tumor is very high: the average life expectancy is 9 to 12months after first diagnosis. The 5-year survival rate from 1986 to 1990was 8.0%. To date, the five-year survival rate following aggressivetherapy including gross tumor resection is still less than 10% (Burtonand Prados, 2000; Nieder et al., 2000; Napolitano et al., 1999; Dazzi etal., 2000). Accordingly, there is a strong medical need for analternative and effective therapeutic method.

Tumor cells of glioblastomas are the most undifferentiated ones amongbrain tumors, so the tumor cells have high potential of migration andproliferation and are highly invasive, leading to very poor prognosis.Glioblastomas lead to death due to rapid, aggressive, and infiltrativegrowth in the brain. The infiltrative growth pattern is responsible forthe unresectable nature of these tumors. Glioblastomas are alsorelatively resistant to radiation and chemotherapy, and, therefore,post-treatment recurrence rates are high. In addition; the immuneresponse to the neoplastic cells is rather ineffective in completelyeradicating all neoplastic cells following resection and radiationtherapy (Roth and Weller, 1999; Dix et al., 1999; Sablotzki et al.,2000).

Glioblastoma is classified into primary glioblastoma (de novo) andsecondary glioblastoma, depending on differences in the gene mechanismduring malignant transformation of undifferentiated astrocytes or glialprecursor cells. Secondary glioblastoma occurs in a younger populationof up to 45 years of age. During 4 to 5 years on average, secondaryglioblastoma develops from lower-grade astrocytoma throughundifferentiated astrocytoma. In contrast, primary glioblastomapredominantly occurs in an older population with a mean age of 55 years.Generally, primary glioblastoma occurs as fulminant glioblastomacharacterized by tumor progression within 3 months from the state withno clinical or pathological abnormalities (Pathology and Genetics of theNervous Systems. 29-39 (IARC Press, Lyon, France, 2000)).

Glioblastoma migrates along myelinated nerves and spreads widely in thecentral nervous system. In most cases surgical treatment shows onlylimited sustainable therapeutic effect (Neurol. Med. Chir. (Tokyo) 34,91-94, 1994; Neurol. Med. Chir. (Tokyo) 33, 425-458, 1993;Neuropathology 17, 186-188, 1997) (Macdonald, 2001; Prados and Levin,2000).

Malignant glioma cells evade detection by the host's immune system byproducing immunosuppressive agents that impair T cell proliferation andproduction of the immune-stimulating cytokine IL-2 (Dix et al., 1999).

Intracranial neoplasms can arise from any of the structures or celltypes present in the CNS, including the brain, meninges, pituitarygland, skull, and even residual embryonic tissue. The overall annualincidence of primary brain tumors in the United States is 14 cases per100,000. The most common primary brain tumors are meningiomas,representing 27% of all primary brain tumors, and glioblastomas,representing 23% of all primary brain tumors (whereas glioblastomasaccount for 40% of malignant brain tumor in adults). Many of thesetumors are aggressive and of high grade. Primary brain tumors are themost common solid tumors in children and the second most frequent causeof cancer death after leukemia in children.

The search for effective treatment of glioblastomas in patients is stillongoing today. Immunotherapy, or treatment via recruitment of the immunesystem, to fight these neoplastic cells has been investigated. Firstencouraging results were obtained in immuno-therapeutic studies inhumans, in which antigen-specific CTL responses could be induced leadingto prolonged median survival times compared to that obtained applyingstandard treatment accompanied by minimal toxicity (Heimberger et al.,2006).

There thus remains a need for a new efficacious and safe treatmentoption for brain tumors and to enhance the well-being of the patientswithout using chemotherapeutic agents or other agents that may lead tosevere side effects. The present invention fulfils this need.

SUMMARY OF THE INVENTION

The present invention provides peptides comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11 or avariant thereof which is 80% homologous to SEQ ID NO: 1 to SEQ ID NO: 11or a variant, which will induce T cells cross-reacting with the peptide.Preferably the peptide maintains the ability to bind to a molecule ofthe human major histocompatibility complex (MHC) class-I or -II andwherein the peptide is capable of stimulating CD4 or CD8 T In otherembodiments, the peptide sequence is at least 80% homologous to thesequence set forth in SEQ D NO:1 to SEQ ID NO:11 and maintains theability to bind to a molecule of the human major histocompatibilitycomplex (MHC) class-I or -II, and wherein the peptide is capable ofstimulating CD4 or CD8 T cells.

In a preferred embodiment the peptide comprises the amino acid sequenceset forth in SEQ ID NO:1. The invention also provides a variant of thispeptide wherein the variant binds to a MHC class I molecule HLA A*0205allele and wherein the variant is capable of stimulating CD8 cells, andwherein the variant has the following motif X₁, X₂, X₃, X₄, X₅, X₆, X₇,X₈, X₉, wherein

X₁ is A, V, or Y;

X₂ is L;

X₃ is T, P, F, I or M;

X₄ is T, E, D, K, or N;

X₅ is L, V, L or I;

X₆ is M, I, V, L, I or A;

X₇ is H or V;

X₈ is Q or Y; and

X₉ is L.

In another preferred embodiment, the peptide comprises the amino acidsequence set forth in SEQ ID NO:2. The invention also provides a variantof this peptide wherein the variant binds to a MHC class I molecule HLAA*02 allele and wherein the variant is capable of stimulating CD8 cells,and wherein the variant has the following motif X₁, X₂, X₃, X₄, X₅, X₆,X₇, X₈, X₉, wherein

X₁ is F, I, L, K, M, Y, or V;

X₂ is L or M;

X₃ is Y, A, F, P, M, S, or R;

X₄ is K, E, G, P, D, or T;

X₅ is V, I, K, Y, N, G, F, or H;

X₆ is I, L, or T;

X₇ is L, A, Y, or H;

X₈ is S, K, E, or S; and

X₉ is L.

In another embodiment, the peptide comprises the amino acid sequence setforth in SEQ ID NO:3. The invention also provides a variant of thispeptide wherein the variant binds to a MHC class I molecule HLA A*02allele and wherein the variant is capable of stimulating CD8 cells, andwherein the variant has the following motif X₁, X₂, X₃, X₄, X₅, X₆, X₇,X₈, X₉, wherein

X₁ is A, I, L, F, K, M, Y or V;

X₂ is I, M or L;

X₃ is I, A, Y, F, P, M, S or R;

X₄ is D, E, G, P or T;

X₅ is G, I, K, Y, N, F or V;

X₆ is V, I, L or T;

X₇ is E, A, Y or H;

X₈ is S, K, or E; and

X₉ is V or L.

The present invention further provides a peptide comprising the aminoacid sequence set forth in SEQ ID NO:4. The invention also provides avariant of this peptide wherein the variant binds to a MHC class Imolecule HLA A*02 allele and wherein the variant is capable ofstimulating CD8 cells, and wherein the variant has the following motifX₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, wherein

X₁ is F, I, L, K, M, Y or V;

X₂ is L or M;

X₃ is L, A, Y, F, P, M, S or R;

X₄ is P, E, G, D, T or K;

X₅ is D, I, K, Y, N, G, F, V or H;

X₆ is T, I or L;

X₇ is D, A, Y or H;

X₈ is G, K, E or S; and

X₉ is L.

In another embodiment, the peptide comprises the amino acid sequence setforth in SEQ ID NO:5. The invention also provides a variant of thispeptide of this peptide wherein the variant binds to a MHC class Imolecule HLA A*02 allele and wherein the variant is capable ofstimulating CD8 cells, and wherein the variant has the following motifX₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, wherein

X₁ is K, I, L, F, M or Y;

X₂ is V, M or L;

X₃ is F, A, Y, P, M or S;

X₄ is A, E, G, P or T;

X₅ is G, I, K, Y, N, F or V;

X₆ is I, L or T;

X₇ is P, A, Y or H;

X₈ is T, K, or E; and

X₉ is V or L.

In another embodiment, the peptide comprises the amino acid sequence setforth in SEQ ID NO:6. The invention also provides a variant of thispeptide wherein the variant binds to a MHC class I molecule HLA A*02allele and wherein the variant is capable of stimulating CD8 cells, andwherein the variant has the following motif X₁, X₂, X₃, X₄, X₅, X₆, X₇,X₈, X₉, wherein

X₁ is Q, V or Y;

X₂ is Q;

X₃ is S, P, F, I or M;

X₄ is D, E, K, N or P;

X₅ is Y, V, L or I;

X₆ is S, I, V, L or A;

X₇ is A, H or V;

X₈ is A or Y; and

X₉ is L.

In another embodiment, the peptide comprises the amino acid sequence setforth in SEQ ID NO:7. The invention also provides a variant of thispeptide wherein the variant binds to a MHC class I molecule HLA A*02allele and wherein the variant is capable of stimulating CD8 cells, andwherein the variant has the following motif X₁, X₂, X₃, X₄, X₅, X₆, X₇,X₈, X₉, wherein

X₁ is T, V or Y;

X₂ is Q;

X₃ is D, P, F, I or M;

X₄ is D, E, K, N or P;

X₅ is Y, V, L or I;

X₆ is V, I, L or A;

X₇ is L, H or V;

X₈ is E or Y; and

X₉ is V or L.

In another embodiment, the peptide comprises the amino acid sequence setforth in SEQ ID NO:8. The invention also provides a variant of thispeptide wherein the variant binds to a MHC class I molecule HLA B*38allele wherein the variant binds to a MHC class I molecule HLA B*38allele and wherein the variant is capable of stimulating CD8 cells, andwherein the variant has the following motif X₁, X₂, X₃, X₄, X₅, X₆, X₇,X₈, X₉, wherein

X₁ is Q or I;

X₂ is H;

X₃ is E or D;

X₄ is G, E, P, L, K or S;

X₅ is T, M, V, A, R, N or H;

X₆ is V, I, T or K;

X₇ is N, Y, V or N;

X₈ is I, K, Y, N or R; and

X₉ is F.

In certain embodiments the peptide has an overall length of between 8and 100, preferably between 8 and 30, and most preferably between 8 and16 amino acids. The peptide preferably has ability to bind to a moleculeof the human major histocompatibility complex (MHC) class-I or -II.

In other embodiments, the peptide consists of or consists essentially ofan amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 11.

The peptides of the invention may be modified or includes non-peptidebonds. The peptides of the invention may be a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii).

The present invention also provides nucleic acids encoding the peptidesof the invention. The nucleic acid may be DNA, cDNA, PNA, CNA, RNA orcombinations thereof. The present invention also provides expressionvectors capable of expressing the nucleic acids of the invention.

Peptides, nucleic acids or expression vectors of the invention may beused in medicine.

The present invention also provides a host cell host cell comprising anucleic acid or an expression vector according to the invention. Inpreferred embodiments, the host cell is an antigen presenting cell, inparticular a dendritic cell.

The present invention further provides a method of producing a peptideof the invention, the method comprising culturing a host cell of theinvention and isolating the peptide from the host cell or its culturemedium.

The present invention also provides an in vitro method for producingactivated cytotoxic T lymphocytes (CTL), the method comprisingcontacting in vitro CTL with antigen loaded human class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor an artificial construct mimicking an antigen-presenting cell for aperiod of time sufficient to activate the CTL in an antigen specificmanner, wherein the antigen is a peptide according to the invention. Ina preferred embodiment, the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor an artificial construct mimicking an antigen-presenting cell bycontacting a sufficient amount of the antigen with an antigen-presentingcell or an artificial construct mimicking an antigen-presenting cell orprecursors thereof. Preferably, the antigen-presenting cell comprises anexpression vector capable of expressing a peptide of the invention.

The present invention further provides activated cytotoxic T lymphocytes(CTL), produced by the methods described herein, which selectivelyrecognise a cell aberrantly expressing a peptide of the invention.

The present invention also provides a method of killing target cells ina patient wherein the target cells aberrantly express a peptide of thepresent invention, the method comprising administering to the patient aneffective number of cytotoxic T lymphocytes (CTL) of the invention.

The present invention also provides the use of any peptide, nucleicacid, expression vector, cell, or activated T lymphocyte of theinvention as a medicament or in the manufacture of a medicament.Preferably the medicament is a vaccine and is active against cancer. Thecancer may be, but is not limited to astrocytoma, pilocytic astrocytoma,dysembryoplastic neuroepithelial tumor, oligodendrogliomas, ependymoma,glioblastoma multiforme, mixed gliomas, oligoastrocytomas,medulloblastoma, retinoblastoma, neuroblastoma, germinoma, teratoma,gangliogliomas, gangliocytoma, central gangliocytoma, primitiveneuroectodermal tumors (PNET, e.g. medulloblastoma, medulloepithelioma,neuroblastoma, retinoblastoma, ependymoblastoma), tumors of the pinealparenchyma (e.g. pineocytoma, pineoblastoma), ependymal cell tumors,choroid plexus tumors, neuroepithelial tumors of uncertain origin (e.g.gliomatosis cerebri, astroblastoma) or glioblastoma cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b show the ESI-liquid chromatography mass spectraidentifying tumor associated peptides (TUMAPs) PTP-001 from glioblastomasample GB1006 and PTP-002 from glioblastoma sample GB6003 that werepresented in a MHC class I restricted manner.

FIG. 2 depicts the mRNA expression profile of the gene PTPRZ1 encodingthe glioblastoma associated peptides shown in Table 1. Expression ofthis gene is absent or very low in normal tissues while it is stronglyincreased in glioblastoma samples (GB 1006T to GB 1011 T; NCH359T andNCH361 T).

FIGS. 3a-3d show a representative example for PTP-002-specific CD8+ Tcells in one healthy HLA-A*0201 donor following in vitro stimulationwith PTP-002 as determined by flow cytometric analysis. CD8+ T cellswere isolated from healthy donor human PBMCs and primed in vitro usingmolecularly defined “artificial antigen presenting cells” (aAPCs) loadedwith co-stimulatory molecules and A*0201/PTP-002 (left diagram) orirrelevant A*0201 peptide (right diagram) (Walter et al., 2003). Afterthree cycles of stimulation, the detection of peptide-reactive cells wasperformed by staining with PTP-002-plus irrelevant peptide tetramers.Cells were gated on CD8+ lymphocyte population and percentages representthe frequencies of tetramer-positive cells within this population.

FIG. 4 shows the affinities of peptides of the invention to HLA-A*0201.Dissociation constants (K_(D)) of the HLA class I peptides and the viralmarker peptide HBV-001 were measured by an ELISA-based assay (seeExample 4).

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has now raised thepossibility of using a host's immune system to foster an immune responsethat is specific for target antigens expressed on the surface of tumorcells, and through this mechanism of action is capable of inducingregression, stasis or slowed growth of the tumor. Various mechanisms ofharnessing both the humoral and cellular arms of the immune system arecurrently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofcytotoxic T cells (CTL) from tumor-infiltrating cell populations or fromperipheral blood suggests that such cells play an important role innatural immune defences against cancer (Cheever et al., 1993; Zeh, IIIet al., 1999). Based on the analysis of 415 specimens from patientssuffering from colorectal cancer, Galon et al. were able to demonstratethat type, density and location of immune cells in tumor tissue areactually a better predictor for survival of patients than the widelyemployed TNM-staging of tumors (Galon et al., 2006). CD8-positive Tcells (TCD8⁺) in particular, which recognise Class I molecules of themajor histocompatibility complex (MHC)-bearing peptides of usually 8 to10 amino acid residues derived from proteins, or defective ribosomalproducts (DRIPs) (Schubert et al., 2000) play an important role in thisresponse. Also, peptides stemming from spliced proteins were describedin the literature. The MHC-molecules of the human are also designated ashuman leukocyte antigens (HLA).

There are two classes of MHC-molecules: MHC class I molecules and MHCclass II molecules. MHC molecules are composed of a alpha heavy chainand beta-2-microglobulin (MHC class I receptors) or an alpha and a betachain (MHC class II receptors), respectively. Their three-dimensionalconformation results in a binding groove, which is used for non-covalentinteraction with peptides. MHC class I molecules can be found on mostcells having a nucleus. MHC class I present peptides that result fromproteolytic cleavage of predominantly endogenous proteins, DRIPs andlarger peptides. MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs during the course of endocytosis, and are subsequently processed(Cresswell, 1994). Complexes of peptide and MHC class I molecules arerecognized by CD8-positive cytotoxic T-lymphocytes bearing theappropriate TCR (T-cell receptor), whereas complexes of peptide and MHCclass II molecules are recognized by CD4-positive-helper-T cells bearingthe appropriate TCR. It is well known that the TCR, the peptide and theMHC are thereby present in a stoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells (Wangand Livingstone, 2003; Sun and Bevan, 2003; Shedlock and Shen, 2003).For this reason the identification of CD4-positive T-cell epitopesderived from tumor associated antigens (TAA) is of great importance forthe development of pharmaceutical products for triggering anti-tumorimmune responses (Kobayashi et al., 2002; Qin et al., 2003; Gnjatic etal., 2003).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave surprisingly been found to express MHC class II molecules (Dengjelet al., 2006).

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CTL effector cells (i.e., CD8-positive T lymphocytes),CD4-positive T cells are sufficient for inhibiting manifestation oftumors via inhibition of angiogenesis by secretion of interferon-gamma(IFNγ) (Qin and Blankenstein, 2000). Additionally, it was shown thatCD4-positive T cells recognizing peptides from tumor-associated antigenspresented by HLA class II molecules can counteract tumor progression viathe induction of antibody (Ab) responses (Kennedy et al., 2003). Incontrast to tumor-associated peptides binding to HLA class I molecules,only a small number of class II ligands of TAA have been described sofar.

Since the constitutive expression of HLA class II molecules is usuallylimited to cells of the immune system (Mach et al., 1996), thepossibility of isolating class II peptides directly from primary tumorswas not considered possible. However, Dengjel et al. were recentlysuccessful in identifying a number of MHC Class II epitopes directlyfrom tumors (WO2007028574, EP1760088 B1); (Dengj el et al., 2006).

For a peptide to trigger (elicit) a cellular immune response, it mustbind to an MHC-molecule. This process is dependent on the allele of theMHC-molecule and specific polymorphisms of the amino acid sequence ofthe peptide. MHC-class-I-binding peptides are usually 8-10 amino acidresidues in length and usually contain two conserved residues(“anchors”) in their sequence that interact with the correspondingbinding groove of the MHC-molecule. In this way each MHC allele has a“binding motif” determining which peptides can bind specifically to thebinding groove (Rammensee et al., 1997).

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they also have to be recognized by T cells bearing specific Tcell receptors (TCR).

The antigens that are recognized by the tumor specific cytotoxic Tlymphocytes, that is, their epitopes, can be molecules derived from allprotein classes, such as enzymes, receptors, transcription factors,etc., which are expressed and, as compared to unaltered cells of thesame origin, up-regulated in cells of the respective tumor.

The current classification of tumor associated antigens comprises thefollowing major groups (Novellino et al., 2005):

1. Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells (van der Bruggen et al., 1991) belong to thisclass, which was originally called cancer-testis (CT) antigens becauseof the expression of its members in histologically different humantumors and, among normal tissues, only in spermatocytes/spermatogonia oftestis and, occasionally, in placenta. Since the cells of testis do notexpress class I and II HLA molecules, these antigens cannot berecognized by T cells in normal tissues and can therefore be consideredas immunologically tumor-specific. Well-known examples for CT antigensare the MAGE family members or NY-ESO-1.

2. Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose; most are found inmelanomas and normal melanocytes. Many of these melanocytelineage-related proteins are involved in the biosynthesis of melanin andare therefore not tumor specific but nevertheless are widely used forcancer immunotherapy. Examples include, but are not limited to,tyrosinase and Melan-A/MART-1 for melanoma or PSA for prostate cancer.

3. Overexpressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir overexpression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, Survivin, Telomerase or WT1.

4. Tumor specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors.

5. TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins that are neither specific nor overexpressedin tumors, but nevertheless become tumor associated by posttranslationalprocesses primarily active in tumors. Examples for this class arise fromaltered glycosylation patterns leading to novel epitopes in tumors, suchas MUC1, or events like protein splicing during degradation, which mayor may not be tumor specific (Hanada et al., 2004; Vigneron et al.,2004).

6. Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

For proteins to be recognized by cytotoxic T-lymphocytes astumor-specific or -associated antigens, and to be used in a therapy,particular prerequisites must be fulfilled. The antigen should beexpressed mainly by tumor cells and not, or in comparably small amounts,by normal healthy tissues. It is furthermore desirable that therespective antigen is not only present in a type of tumor, but also inhigh concentrations (i.e. copy numbers of the respective peptide percell). Tumor-specific and tumor-associated antigens are often derivedfrom proteins directly involved in transformation of a normal cell to atumor cell due to a function e.g. in cell cycle control or suppressionof apoptosis. Additionally, also downstream targets of the proteinsdirectly causative for a transformation may be upregulated and thus maybe indirectly tumor-associated. Such indirectly tumor-associatedantigens may also be targets of a vaccination approach (Singh-Jasuja etat, 2004). In both cases it is essential that epitopes are present inthe amino acid sequence of the antigen, since such a peptide(“immunogenic peptide”) that is derived from a tumor associated antigenshould lead to an in vitro or in vivo T-cell-response.

Basically, any peptide able to bind a MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell with a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a tumorvaccine. The methods for identifying and characterizing the TAAs arebased on the use of CTL that can be isolated from patients or healthysubjects, or they are based on the generation of differentialtranscription profiles or differential peptide expression patternsbetween tumors and normal tissues (Lemmel et al., 2004; Weinschenk etal., 2002).

However, the identification of genes over-expressed in tumor tissues orhuman tumor cell lines, or selectively expressed in such tissues or celllines, does not provide precise information as to the use of theantigens transcribed from these genes in an immune therapy. This isbecause only an individual subpopulation of epitopes of these antigensare suitable for such an application since a T cell with a correspondingTCR has to be present and immunological tolerance for this particularepitope needs to be absent or minimal. It is therefore important toselect only those peptides from over-expressed or selectively expressedproteins that are presented in connection with MHC molecules againstwhich a functional T cell can be found. Such a functional T cell isdefined as a T cell, which upon stimulation with a specific antigen, canbe clonally expanded and is able to execute effector functions(“effector T cell”). Typical effector functions of T cells include thesecretion of Interferon-gamma, perforin, and granzymes.

T-helper cells play an important role in orchestrating the effectorfunction of CTLs in anti-tumor immunity. T-helper cell epitopes thattrigger a T-helper cell response of the T_(H1) type support effectorfunctions of CD8-positive killer T cells, which include cytotoxicfunctions directed against tumor cells displaying tumor-associatedpeptide/MHC complexes on their cell surfaces. In this waytumor-associated T-helper cell peptide epitopes, alone or in combinationwith other tumor-associated peptides, can serve as active pharmaceuticalingredients of vaccine compositions that stimulate anti-tumor immuneresponses.

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+CTLs (ligand: MHC class I molecule+peptide epitope) or by CD4-positiveT-helper cells (ligand: MHC class II molecule+peptide epitope) isimportant in the development of tumor vaccines. It is therefore anobject of the present invention, to provide novel amino acid sequencesfor peptides that are able to bind to MHC complexes of either class.

Accordingly, the present invention provides peptides that are useful intreating glioblastoma. These peptides were directly shown by massspectrometry to be naturally presented by HLA molecules on primary humanglioblastoma samples (see example 1 and FIGS. 1a and 1b ). The sourcegene from which these peptides are derived—PTPRZ1—was shown to be highlyoverexpressed in glioblastoma compared with normal tissues (see example2 and FIG. 2) demonstrating a high degree of tumor association of thesepeptides, i.e. these peptides are strongly presented on tumor tissue butnot on normal tissues. HLA-bound peptides can be recognized by theimmune system, specifically T lymphocytes/T cells. T cells can destroythe cells presenting the recognized HLA/peptide complex, e.g.glioblastoma tumor cells presenting the PTPRZ1-derived peptides. Severalpeptides of the present invention have been shown to be capable ofstimulating T cell responses (see example 3 and FIGS. 3a-3d ). Thus, thepeptides are useful for generating an immune response in a patient bywhich tumor cells can be destroyed. An immune response in a patient canbe induced by direct administration of the described peptides orsuitable precursor substances (e.g. elongated peptides, proteins, ornucleic acids encoding these peptides) to the patient, ideally incombination with an agent enhancing the immunogenicity (i.e. anadjuvant). The immune response originating from such a therapeuticvaccination can be expected to be highly specific against tumor cellsbecause the target peptides of the present invention are not presentedon normal tissues, preventing the risk of undesired autoimmune reactionsagainst normal cells in the patient.

The presence of claimed tumor associated peptides (TUMAPs) on tissuebiopsies can assist a pathologist in diagnosis of cancer. Detection ofcertain TUMAPs by means of antibodies, mass spectrometry or othermethods known in the art can tell the pathologist that the tissue ismalignant or inflamed or generally diseased. Presence of groups ofTUMAPs can enable classification or subclassification of diseasedtissues.

The detection of TUMAPs on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmunosurveillance. Thus, presence of TUMAPs shows that this mechanismis not exploited by the analyzed cells.

TUMAPs might be used to analyze lymphocyte responses against thoseTUMAPs such as T cell responses or antibody responses against the TUMAPor the TUMAP complexed to MHC molecules. These lymphocyte responses canbe used as prognostic markers for decision on further therapy steps.These responses can also be used as surrogate markers in immunotherapyapproaches aiming to induce lymphocyte responses by different means,e.g. vaccination of protein, nucleic acids, autologous materials,adoptive transfer of lymphocytes. In gene therapy settings, lymphocyteresponses against TUMAPs can be considered in the assessment of sideeffects. Monitoring of lymphocyte responses might also be a valuabletool for follow-up examinations of transplantation therapies, e.g. forthe detection of graft versus host and host versus graft diseases.

TUMAPs can be used to generate and develop specific antibodies againstMHC/TUMAP complexes. These can be used for therapy, targeting toxins orradioactive substances to the diseased tissue. Another use of theseantibodies can be targeting radionuclides to the diseased tissue forimaging purposes such as PET. This use can help to detect smallmetastases or to determine the size and precise localization of diseasedtissues.

Table 1 shows the peptides according to the invention, their respectiveSEQ ID NO:, the HLA alleles to which the respective peptides bind, andthe source proteins from which these peptides may arise.

TABLE 1 Peptides of the present invention SEQ ID Peptide Source NO CodeSequence HLA Alleles Protein(s)  1 PTP-001 ALTTLMHQL A*0205 PTPRZ1  2PTP-002 FLYKVILSL A*02 PTPRZ1  3 PTP-003 AIIDGVESV A*02 PTPRZ1  4PTP-004 FLLPDTDGL A*02 PTPRZ1  5 PTP-005 KVFAGIPTV A*02 PTPRZ1  6PTP-006 QQSDYSAAL A*02# PTPRZ1  7 PTP-007 TQDDYVLEV A*02# PTPRZ1, PTPRG 8 PTP-008 QHEGTVNIF B*38 PTPRZ1  9 PTP-009 SVFGDDNKALSK not determinedPTPRZ1 10 PTP-010 EIGWSYTGALNQKN HLA-DR PTPRZ1 11 CHI-001 SLWAGVVVL A*02CHI3L2 #probably subtype A*205

Surprisingly, PTP-002, SEQ ID NO:2 was also found to be presented inprimary adenousquamous carcinoma (a form of lung cancer) and cantherefore also be used for treating this type of cancer.

Protein Tyrosine Phosphatase, Receptor-Type, Zeta1 (PTPRZ1, PTP-ξ)

PTPRZ1 is a member of the receptor type protein tyrosine phosphatasefamily and encodes a single-pass type I membrane protein with twocytoplasmic tyrosine-protein phosphatase domains, an alpha-carbonicanhydrase domain and a fibronectin type-III domain (Wu et al., 2006), inbreast cancer (Perez-Pinera et al., 2007), in the remyelinatingoligodendrocytes of multiple sclerosis lesions (Harroch et al., 2002),and in human embryonic kidney cells under hypoxic conditions (Wang etal., 2005).

Both the protein and transcript are overexpressed in glioblastoma cells,promoting their haptotactic migration (Lu et al., 2005). Furthermore,PTRPZ1 is frequently amplified at the genomic DNA level in glioblastoma(Mulholland et al., 2006).

Kaplan et al. cloned 3 human receptor PTP genes, including PTP-γ (Kaplanet al., 1990). It was shown that one PTPG allele was lost in 3 of 5renal carcinoma cell lines and in 5 of 10 lung carcinoma tumor samplestested. PTP-γ mRNA was expressed in kidney cell lines and lung celllines but not in several hematopoietic cell lines tested. Thus, thePTP-γ gene appeared to have characteristics suggesting that it may be atumor suppressor gene in renal and lung carcinoma. Gebbink et al.isolated a mouse cDNA of 5.7 kb, encoding a ‘new’ member of the familyof receptor-like protein-tyrosine phosphatases, termed RPTPμ (Gebbink etal., 1991). The cDNA predicted a protein of 1,432 amino acids (notincluding the signal peptide) with a calculated molecular mass of161,636 Da. In addition, they cloned the human homolog, which showed98.7% amino acid homology to the mouse protein. The predicted mouseprotein consisted of a 722-amino acid extracellular region, containing13 potential N-glycosylation sites, a single transmembrane domain, and a688-amino acid intracellular part containing two tandem repeatshomologous to the catalytic domains of other tyrosine phosphatases. RNAblot analysis showed a single transcript that was most abundant in lungbut present in much lower amounts in brain and heart as well. The humanPTPμ gene was assigned to 18pter-q11 by Southern analysis ofhuman/rodent somatic cell hybrid clones.

PTP-ε cDNA was isolated by Krueger et al. (Krueger et al., 1990). The700-amino acid protein has a short extracellular domain and two tandemlyrepeated intracellular PTPase domains. High levels of PTP-etranscription were noted in the mouse brain and testes. Both isoforms ofPTP-ε-transmembrane, receptor-type isoform and a shorter, cytoplasmicisoform—appear to arise from a single gene through the use ofalternative promoters and 5-prime exons.

Barnea et al. (Barnea et al., 1993) cloned cDNAs for the human and mousePTP-γ gene (designated PTP-γ by that group) from brain cDNA libraries,and analyzed their predicted polypeptide sequences. The human(1,445-amino acid) and mouse (1,442-amino acid) sequences share 95%identity at the amino acid level and predict a putative extracellulardomain, a single transmembrane domain, and a cytoplasmic region with 2tandem catalytic tyrosine phosphatase domains. The extracellular domaincontains a stretch of 266 amino acids that are highly similar to thezinc-containing enzyme carbonic anhydrase (MIM 114800), suggesting thatPTP-γ and PTP-ξ (PTPRZ1) represent a subfamily of 25 receptor tyrosinephosphatases. The gene for PTP-γ has 30 exons and is approximately 780kb in size. It is much larger than the other receptor PTP genes, withthe CD45 gene (MIM 151460) around 100 kb and the others even smaller.

Another receptor-type tyrosine phosphatase, protein tyrosine phosphatasezeta (PTPRZ1) [also known as PTP-ξ, HPTP-ZETA, HPTPZ, RPTP-BETA(β), orRPTPB] was isolated as a cDNA sequence by two groups in the earlynineties. Levy et al. (Levy et al., 1993) isolated cDNA clones from ahuman infant brainstem mRNA expression library, and deduced the completeamino acid sequence of a large receptor-type protein tyrosinephosphatase containing 2,307 amino acids.

Levy found that the protein, which they designated PTPβ (PTPRZ1), is atransmembrane protein with 2 cytoplasmic PTPase domains and a1,616-amino acid extracellular domain. As in PTP-γ (MIM 176886), the 266N-terminal residues of the extracellular domain have a high degree ofsimilarity to carbonic anhydrases (see MIM 114880). The human geneencoding PTPRZ1 has been mapped to chromosome 7q31.3-q32 by chromosomalin situ hybridization (Ariyama et al., 1995). Northern blot analysis hasshown that showed that PTP-zeta is expressed only in the human centralnervous system. By in situ hybridization, (Levy et al., 1993) localizedthe expression to different regions of the adult human brain, includingthe Purkinje cell layer of the cerebellum, the dentate gyrus, and thesubependymal layer of the anterior horn of the lateral ventricle. Levystated that this was the first mammalian tyrosine phosphatase whoseexpression is restricted to the nervous system. In addition, high levelsof expression in the murine embryonic brain suggested an important rolein CNS development.

Thus, the PTP receptor family of proteins has been characterized as afairly diverse family of membrane-bound receptors, and non-membranebound isoforms, which share a common PTPase cytosol domain architecture.Although their expression in fetal and embryonic tissues has suggested adevelopmental biology role for the proteins, their full function innormal and disease state biology is still not fully understood.

U.S. Pat. No. 6,455,026 identified PTP-ξ (PTPRZ1) as a target in thetreatment and visualization of brain tumors. The application providedmethods and reagents for specifically targeting brain tumor cells forboth therapeutic and imaging purposes. PTP-ξ affinity-based compoundsand compositions useful in treating a brain tumor in a patient wereprovided, whereas the compositions and compounds generally fell into twogroups: PTP-ξ-binding conjugate compounds, which comprise a cytotoxicmoiety, which inhibits the growth of tumor cells; and PTP-ξ-bindingcompound compositions in which the PTP-ξ binding moiety alters thenormal function of PTP-ξ in the tumor cell, thus inhibiting cell growth.

In a first group, PTP ξ-binding therapeutic conjugate compounds wereprovided. These compounds had the general formula α(P_(z))C, whereinα(P_(z)) were one or more moieties that specifically bound to a humanprotein tyrosine phosphatase-ξ, and C was one or more cytotoxicmoieties. In preferred embodiments (which was disclosed for all groups)α(P_(z)) was disclosed to be an antibody or an antibody fragment. In asecond group, PTP-ξ-binding therapeutic compounds were provided thataltered the normal function of PTP-ξ in brain tumor cells and inhibitedbrain tumor cell growth. These PTPRZ1-binding therapeutic compounds hadthe general formula α(P_(z)) wherein α(P_(z)) were one or more moietiesthat specifically bound to a human protein tyrosine phosphatase-ξ, andwherein the binding of α(P_(z)) altered the function of protein tyrosinephosphatase-ξ.

U.S. Pat. No. 7,060,275 B2 discloses splicing variants of PTPRZ1,vectors including these variants and antigens against various variants.

Chitinase 3-Like 2 (CHI3L2)

CHI3L2 was originally identified from chondrocytes. It has beenfrequently described as a target antigen in rheumatoid arthritis. Norelevant association of CHI3L2 with cancer was identified. Chitinase3-like proteins have been implied in stimulating proliferation of humanconnective tissue cells, e.g. fibroblasts, by activating extracellularsignal-regulated kinase and PKB mediated signalling pathways (Recklies AD, et al., 2002). In mice, chitinase 3-like proteins have been found tobe strongly upregulated in Helicobacter-induced gastric cancer models(Takaishi S, et al., 2007).

Nowhere in the prior art has the use of MHC-binding peptides derivedfrom PTPRZ1 or CHI2L2 as active pharmaceutical ingredients for thetreatment of brain tumors been considered.

Accordingly, in a first aspect, the invention provides a peptidecomprising a sequence that is selected from the group of SEQ ID NO: 1 toSEQ ID NO: 11 or a variant thereof that is 80% homologous to SEQ ID NO:1 to SEQ ID NO: 11 or a variant that will induce T cells cross-reactingwith the peptide.

The peptides of the invention have the ability to bind to a molecule ofthe human major histocompatibility complex (MHC) class-I or -II.

In the present invention, the term “homologous” refers to the degree ofidentity between sequences of two amino acid sequences, i.e. peptide orpolypeptide sequences. The aforementioned “homology” is determined bycomparing two sequences aligned under optimal conditions over thesequences to be compared. The sequences to be compared may have anaddition or deletion (for example, gap and the like) in the optimumalignment of the two sequences. Such a sequence homology can becalculated by creating an alignment using, for example, the ClustalWalgorithm (Nucleic Acid Res., 22(22): 4673 4680 (1994). Commonlyavailable sequence analysis software, more specifically, Vector NTI,GENETYX or analysis tools provided by public databases may be used forsequence alignment.

A person skilled in the art will be able to assess whether T cellsinduced by a variant of a specific peptide can cross-react with thepeptide itself (Fong et al., 2001b); (Zaremba et al., 1997; Colombettiet al., 2006; Appay et al., 2006).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)so that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in SEQ ID NO:1-11. For example, a peptide may be modifiedso that it at least maintains, if not improves, the ability to interactwith, and bind to the binding groove of, a suitable MHC molecule, suchas HLA-A*02 or -DR, and in that way it at least maintains, if notimproves, the ability to bind to the TCR of activated CTL. These CTL cansubsequently cross-react with cells and kill cells that express apolypeptide containing the natural amino acid sequence of the cognatepeptide as defined in the aspects of the invention. As can be derivedfrom the scientific literature (Rammensee et al., 1997) and databases(Rammensee et al., 1999), certain positions of HLA binding peptides aretypically anchor residues forming a core sequence fitting to the bindingmotif of the HLA receptor, which is defined by polar, electrophysical,hydrophobic and spatial properties of the polypeptide chainsconstituting the binding groove. Thus one skilled in the art would beable to modify the amino acid sequences set forth in SEQ ID NO:1-11, bymaintaining the known anchor residues, and would be able to determinewhether such variants maintain the ability to bind MHC class I or IImolecules. The variants of the present invention retain the ability tobind to the TCR of activated CTL, which can subsequently cross-reactwith- and kill cells that express a polypeptide containing the naturalamino acid sequence of the cognate peptide as defined in the aspects ofthe invention.

Those amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith another amino acid whose incorporation does not substantiallyaffect T-cell reactivity and does not eliminate binding to the relevantMHC. Thus, apart from the proviso given, the peptide of the inventionmay be any peptide (by which term the inventors include oligopeptide orpolypeptide) that includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 2 Variants and motif of the peptides according to SEQ ID NO: 1 to11 Position 1 2 3 4 5 6 7 8 9 PTP-001 Peptide Code A L T T L M H Q LVariants V V P E V I H Y Y I F D L V V M I K I L M N A P PTP-002 PeptideCode F L Y K V I L S L Variants M E K I A G I I A E L Y P K L Y S F F DY T H K P T N M M G Y S F V R V K H PTP-003 Peptide Code A I I D G V E SV Variants M L L L E K I A G I I A E L Y P K L Y F F T Y T H K P N M M FY S V V R PTP-004 Peptide Code F L L P D T D G L Variants M L E K I A GI I A E L Y D K L Y S K F T Y H M P N Y M G V S F R V K H PTP-005Peptide Code K V F A G I P T V Variants M L L L E K I A G I A E L Y P KL Y F P T Y T H M M N Y S F V PTP-006 Peptide Code Q Q S D Y S A A LVariants V P E V I H Y Y F K L V V I N I L M P A PTP-007 Peptide Code TQ D D Y V L E V Variants V P E V I H Y L Y F K L V V I N I L M P APTP-008 Peptide Code Q H E G T V N I F Variants D I E M V Y K P V I V YL A T N N E R K R G N L H K S CHI-001 Peptide Code S L W A G V V V LVariants M E K I A G I I A E L Y P K L Y S F F D Y T H K P T N M M Y S FV R V K H

It is furthermore known for MHC-class II presented peptides that thesepeptides are composed of a “core sequence” having a amino acid sequencefitting to a certain HLA-allele-specific motif and, optionally, N-and/or C-terminal extensions that do not interfere with the function ofthe core sequence (i.e. are deemed as irrelevant for the interaction ofthe peptide and all or a subset of T cell clones recognising the naturalcounterpart). The N- and/or C-terminal extensions can, for example, bebetween 1 to 10 amino acids in length, respectively. These peptides canbe used either directly to load MHC class II molecules or the sequencecan be cloned into the vectors according to the description hereinbelow. As these peptides constitute the final product of the processingof larger peptides within the cell, longer peptides can be used as well.The peptides of the invention may be of any size, but typically they maybe less than 100,000 in molecular weight, preferably less than 50,000,more preferably less than 10,000 and typically about 5,000. In terms ofthe number of amino acid residues, the peptides of the invention mayhave fewer than 1,000 residues, preferably fewer than 500 residues, morepreferably fewer than 100, more preferably fewer than 100 and mostpreferably between 30 and 8 residues. Accordingly, the present inventionalso provides peptides and variants thereof wherein the peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 16, namely 8, 9, 10, 11, 12,13, 14, 15, or 16 amino acids.

Correspondingly, naturally occurring or artificial variants that induceT cells cross-reacting with a peptide of the invention are often lengthvariants. Examples for such naturally occurring length variants aregiven in Table 1 for SEQ ID NO: 11.

If a peptide that is longer than around 12 amino acid residues is useddirectly to bind to a MHC class H molecule, it is preferred that theresidues flanking the core HLA binding region do not substantiallyaffect the ability of the peptide to bind specifically to the bindinggroove of the MHC class II molecule or to present the peptide to the T(-helper) cell. However, as already indicated above, it will beappreciated that larger peptides may be used, e.g. when encoded by apolynucleotide, since these larger peptides may be fragmented bysuitable antigen-presenting cells.

It is also possible, that MHC class I epitopes, although usually between8-10 amino acids long, are generated by peptide processing from longerpeptides or proteins that include the actual epitope. It is preferredthat the residues flanking the actual epitope do not substantiallyaffect proteolytic cleavage necessary to expose the actual epitopeduring processing.

Accordingly, the present invention also provides peptides and variantsof MHC class epitopes wherein the peptide or variant has an overalllength of between 8 and 100, preferably between 8 and 30, and mostpreferred between 8 and 16, namely 8, 9, 10, 11, 12, 13, 14, 15, or 16amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art,for example those described in the literature for different MHC class IIalleles (e.g., Vogt et al., 1994; Malcherek et al., 1994; Manici et al.,1999; Hammer et al., 1995; Tompkins et al., 1993; Boyton et al., 1998).

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 11.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO: 11 or a variant thereof, contains additional N-and/or C-terminally located stretches of amino acids do not necessarilyform part of the peptide that functions as an epitope for MHC moleculeepitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. Accordingly, in one embodiment of the present invention, thepeptide is a fusion protein that comprises, for example, the 80N-terminal amino acids of the HLA-DR antigen-associated invariant chain(p33, in the following “Ii”) as derived from the NCBI, GenBankAccession-number X004.97 (Strubin, M. et al. 1984).

Examples of preferred peptides having a specific HLA-subtype and capableof stimulating CD8 cells, are peptides that comprise the specific anchoramino acid-motif as depicted in the following table 2a:

TABLE 2A HLA-subtypes and anchor motifs of preferred peptides HLA-Position Peptide subtype 1 2 3 4 5 6 7 8 9 1 A*0205 Peptide Code A L T TL M H Q L Anchor x L x x x x x x L motif 2 A*02 Peptide Code F L Y K V IL S L Anchor x L x x x x x x L motif 3 A*02 Peptide Code A I I D G V E SV Anchor x I x x x x x x V motif 4 A*02 Peptide Code F L L P D T D G LAnchor x L x x x x x x L motif 5 A*02 Peptide Code K V F A G I P T VAnchor x V x x x x x x V motif 6 A*02 Peptide Code Q Q S D Y S A A L(probably subtype A*205) Anchor x Q x x x x x x L motif 7 A*02 PeptideCode T Q D D Y V L E V (probably subtype A*205) Anchor x Q x x x x x x Vmotif 8 B*38 Peptide Code Q H E G T V N I F Anchor x H E x x x x x Fmotif 11  A*02 Peptide Code S L W A G V V V L x L x x x x x x L X A*02General anchor motif x L/I/V x x x x x x L/V for peptides

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules to elicit a stronger immuneresponse. Methods for these types of optimisation of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide, bond amino acid residues are not joined by peptide(—CO—NH—) linkages, but rather the peptide bond is reversed. Suchretro-inverso peptidomimetics may be made using methods known in theart, for example such as those described in Meziere et al. (1997) J.Immunol. 159, 3230-3237, incorporated herein by reference. This approachinvolves making pseudopeptides containing changes involving thebackbone, and not the orientation of side chains. Meziere et al. (1997)show that for MHC binding and T helper cell responses, thesepseudopeptides are useful. Retro-inverse peptides, containing NH—CObonds instead of CO—NH peptide bonds are much more resistant toproteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains that involves polypeptides synthesised by standardprocedures and the non-peptide bond synthesised by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance, for example, the stability, bioavailability, and/oraffinity of the peptides. For example, hydrophobic groups such ascarbobenzoxyl, dansyl, or t-butyloxycarbonyl groups may be added to thepeptides' amino termini. Likewise, an acetyl group or a9-fluorenylmethoxy-carbonyl group may be placed at the peptides' aminotermini. Additionally, the hydrophobic group, t-butyloxycarbonyl, or anamido group may be added to the peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2005, which is incorporatedherein by reference. Chemical modification of amino acids includes, butis not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley & Sons NY 1995-2000) for more extensivemethodology relating to chemical modification of proteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich provide information onspecific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. Diethylpyrocarbonate is a reagent for themodification of histidyl residues in proteins. Histidine can also bemodified using 4-hydroxy-2-nonenal. The reaction of lysine residues andother α-amino groups is, for example, useful in binding of peptides tosurfaces or the cross-linking of proteins/peptides. Lysine is the siteof attachment of poly(ethylene)glycol and the major site of modificationin the glycation of proteins. Methionine residues in proteins can bemodified with e.g. iodoacetamide, bromoethylamine, chloramine T.Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions. Recent studies onthe modification of tryptophan have used N-bromosuccinimide,2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life, whilecross-linking of proteins with glutaraldehyde, polyethyleneglycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesised by the Fmoc-polyimidemode of solid-phase peptide synthesis as disclosed by Lu et al. (1981)and references therein. Temporary N-amino group protection is affordedby the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage ofthis highly base-labile protecting group is done using 20% piperidine inN, N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalisingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used areethandithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, Bruckdorfer et al.2004 and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure thatupon lyophilisation of the aqueous phase affords the crude peptide freeof scavengers. Reagents for peptide synthesis are generally availablefrom e.g. Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitril/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

A further aspect of the invention provides a nucleic acid (e.g.polynucleotide) encoding a peptide or variant of the invention. Thepolynucleotide may be e.g. DNA, cDNA, PNA, CNA, RNA or combinationsthereof, either single- and/or double-stranded, or native or stabilizedforms of polynucleotides, such as e.g. polynucleotides with aphosphorothioate backbone, and it may or may not contain introns so longas it codes for the peptide. Of course, only peptides containingnaturally occurring amino acid residues joined by naturally occurringpeptide bonds are encodable by a polynucleotide. A still further aspectof the invention provides an expression vector capable of expressing apolypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc, New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention utilizes the polymerase chain reaction as disclosed by (Saikiet al. (1988)). This method may be used for introducing the DNA into asuitable vector, for example by engineering in suitable restrictionsites, or it may be used to modify the DNA in other useful ways as isknown in the art.

If viral vectors are used, pox- or adenovirus vectors are preferred. TheDNA (or in the case of retroviral vectors, RNA) may then be expressed ina suitable host to produce a polypeptide comprising the peptide orvariant of the invention. Thus, the DNA encoding the peptide or variantof the invention may be used in accordance with known techniques,appropriately modified in view of the teachings contained herein, toconstruct an expression vector, which is then used to transform anappropriate host cell for the expression and production of thepolypeptide of the invention. Such techniques include those disclosed inU.S. Pat. No. 4,440,859 issued 3 Apr. 1984 to Rutter et al., U.S. Pat.No. 4,530,901 issued 23 Jul. 1985 to Weissman, U.S. Pat. No. 4,582,800issued 15 Apr. 1986 to Crowl, U.S. Pat. No. 4,677,063 issued 30 Jun.1987 to Mark et al., U.S. Pat. No. 4,678,751 issued 7 Jul. 1987 toGoeddel, U.S. Pat. No. 4,704,362 issued 3 Nov. 1987 to Itakura et al.,U.S. Pat. No. 4,710,463 issued 1 Dec. 1987 to Murray, U.S. Pat. No.4,757,006 issued 12 Jul. 1988 to Toole, Jr. et al., U.S. Pat. No.4,766,075 issued 23 Aug. 1988 to Goeddel et al. and 4,810,648 issued 7Mar. 1989 to Stalker, all of which are incorporated herein by reference.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the preprotrypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501 which aregenerally available from Stratagene Cloning Systems, La Jolla, Calif.92037, USA. Preferred mammalian host cells include Chinese hamster ovary(CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryocells NIH/3T3 available from the ATCC as CRL 1658, monkey kidney-derivedCOS-1 cells available from the ATCC as CRL 1650 and 293 cells which arehuman embryonic kidney cells. Preferred insect cells are Sf9 cells whichcan be transfected with baculovirus expression vectors. An overviewregarding the choice of suitable host cells for expression can be foundin, for example, the textbook of Paulina Balbás and Argelia Lorence“Methods in Molecular Biology Recombinant Gene Expression, Reviews andProtocols,” Part One, Second Edition, ISBN 978-1-58829-262-9, and otherliterature known to the person of skill.

Transformation of appropriate cell, hosts with a DNA construct of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (1972) Proc.Natl. Acad. Sci. USA 69, 2110 and Sambrook et al. (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. Transformation of yeast cells is described in Sherman etal. (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold SpringHarbor, N.Y. The method of Beggs (1978) Nature 275, 104-109 is alsouseful. With regard to vertebrate cells, reagents useful in transfectingsuch cells, for example calcium phosphate and DEAE-dextran or liposomeformulations, are available from Stratagene Cloning Systems, or LifeTechnologies Inc., Gaithersburg, Md. 20877, USA. Electroporation is alsouseful for transforming and/or transfecting cells and is well known inthe art for transforming yeast cell, bacterial cells, insect cells andvertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may be used to express the peptides ofthe invention such that they may be loaded into appropriate MHCmolecules. Thus, the present invention provides a host cell comprising anucleic acid or an expression vector according to the invention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) are currently under investigation for the treatment of prostatecancer (Sipuleucel-T) (Small E J et al. 2006; Rini et al. 2006).

A further aspect of the invention provides a method of producing apeptide or its variant. The method comprises culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of between 50 μg and 1.5 mg, preferably 125 μg to500 μs, of peptide or DNA may be given and will depend on the respectivepeptide or DNA. Doses of this range were successfully used in previoustrials (Brunsvig et al. 2006; Staehler et al. 2007).

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human class I or II MHC molecules expressed onthe surface of a suitable antigen-presenting cell for a period of timesufficient to activate the T cell in an antigen specific manner. Theantigen is a peptide according to the invention. Preferably a sufficientamount of the antigen is used with an antigen-presenting cell.

In the case of a MHC class II epitope used as an antigen, the T cellsare CD4-positive helper cells, preferably of T_(H1)-type. The MHC classII molecules may be expressed on the surface of any suitable cell.Preferably the cell does not naturally express MHC class II molecules(in which case the cell has been transfected to express such a molecule.Alternatively, if the cell naturally expresses MHC class II molecules,it is preferred that it is defective in the antigen-processing orantigen-presenting pathways. In this way, it is possible for the cellexpressing the MHC class II molecule to be completely loaded with achosen peptide antigen before activating the T cell.

The antigen-presenting cell (or stimulator cell) typically has MHC classII molecules on its surface and preferably is itself substantiallyincapable of loading the MHC class II molecule with the selectedantigen. The MHC class II molecule may readily be loaded with theselected antigen in vitro.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is theTransporter associated with Antigen Processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Karre et al. 1985.

The host cell preferably expresses substantially no MHC class Imolecules before transfection. If the stimulator cell expresses a MHCmolecule it is preferred that that the molecule is important forproviding a co-stimulatory signal for T-cells such as any of B7.1, B7.2,ICAM-1 and LFA 3.

The nucleic acid sequences of numerous MHC class II molecules, and ofthe costimulator molecules, are publicly available from the GenBank andEMBL databases.

Similarly, in when a MHC class I epitope is used as an antigen, the Tcells are CD8-positive CTLs.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 11 or its variant aminoacid sequence.

A number of other methods may be used for generating CTL in vitro. Forexample, the methods described in Peoples et al. (1995) and Kawakami etal. (1992) use autologous tumor-infiltrating lymphocytes in thegeneration of CTL. Plebanski et al. (1995) makes use of autologousperipheral blood lymphocytes (PLBs) in the preparation of CTL. Jochmuset al. (1997) describes the production of autologous CTL by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus. Hill et al. (1995) and Jerome et al. (1993) employ Bcells in the production of autologous CTL. In addition, macrophagespulsed with peptide or polypeptide, or infected with recombinant virus,may be used in the preparation of autologous CTL. S. Walter et al.(2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable method forgenerating T cells against the peptide of choice. In this study, aAPCswere generated by the coupling of preformed MHC:peptide complexes to thesurface of polystyrene particles (microbeads) by biotin:streptavidinbiochemistry. This system permits the exact control of the MHC densityon aAPCs, which allows one to selectively elicit high- or low-avidityantigen-specific T cell responses with high efficiency from bloodsamples. Apart from MHC:peptide complexes, aAPCs should carry otherproteins with co-stimulatory activity like anti-CD28 antibodies coupledto their surface. Furthermore such aAPC-based system often requires theaddition of appropriate soluble factors, e. g. cytokines likeinterleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition plant viruses may be used. See for example,Porta et al (1994), which describes the development of cowpea mosaicvirus as a high-yielding system for the presentation of foreignpeptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells that are produced by the above method will selectivelyrecognize a cell that aberrantly expresses a polypeptide that comprisesan amino acid sequence of SEQ ID NO: 1 to 11.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease which can be readily tested for anddetected.

In vivo, the target cells for the CD4-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the invention may be used as active ingredients of atherapeutic composition. Thus, the invention also provides a method ofkilling target cells in a patient whose target cells aberrantly expressa polypeptide comprising an amino acid sequence of the invention. Themethod comprises administering to the patient an effective number of Tcells as defined above.

By “aberrantly expressed” it is meant that the polypeptide isover-expressed compared to normal levels of expression or that the geneis silent in the tissue from which the tumor is derived but in the tumorit is expressed. By “over-expressed” it is meant that the polypeptide ispresent at a level at least 1.2× that present in normal tissue;preferably at least 2× and more preferably at least 5× or 10× the levelpresent in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art and can be found, e.g. in (Rosenberg et al., 1987; Rosenberget al., 1988; Dudley et al., 2002; Yee et al., 2002; Dudley et al.,2005); reviewed in (Gattinoni et al., 2006) and (Morgan et al., 2006).

Any molecule of the invention, i.e. the peptide, nucleic acid,expression vector, cell, activated CTL, T-cell receptor or the nucleicacid encoding it is useful for the treatment of disorders characterisedby cells escaping an immune response. Therefore any molecule of thepresent invention may be used as medicament or in the manufacture of amedicament. The molecule may be used by itself or combined with othermolecule(s) of the invention or (a) known molecule(s).

Preferably the medicament of the present invention is a vaccine. It maybe administered directly into the patient, into the affected organ orsystemically i.d., i.m., s.c., i.p. and i.v., or applied ex vivo tocells derived from the patient or a human cell line, which aresubsequently administered to the patient, or used in vitro to select asubpopulation of immune cells derived from the patient, which are thenre-administered to the patient. If the nucleic acid is administered tocells in vitro, it may be useful for the cells to be transfected so asto co-express immune-stimulating cytokines, such as interleukin-2. Thepeptide may be substantially pure, or combined with animmune-stimulating adjuvant (see below) or used in combination withimmune-stimulatory cytokines, or be administered with a suitabledelivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and Longenecker et al. (1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 CTLs is more efficient in the presence of CD4 T-helper cells. Thus,for MHC Class I epitopes that stimulate CD8 CTL the fusion partner orsections of a hybrid molecule suitably provide epitopes that stimulateCD4-positive T cells. CD4- and CD8-stimulating epitopes are well knownin the art and include those identified in the present invention.

In one aspect the vaccine comprises at least one peptide, preferably twoto 50, more preferably two to 25, even more preferably two to 15 andmost preferably two, three, four, five, six, seven, eight, nine, ten,eleven, twelve or thirteen peptides. The peptide(s) may be derived fromone or more specific TAAs and may bind to MHC class I and/or class IImolecules.

Preferably when the peptides of the invention are used in a vaccine ormedicament of the invention, they are present as a salt, such as forexample, but not limited to an acetate salt or a chloride salt.

The polynucleotide may be substantially pure, or contained in a suitablevector or delivery system. The nucleic acid may be DNA, cDNA, PNA, CNA,RNA or a combination thereof.

Methods for designing and introducing such a nucleic acid are well knownin the art. An overview is provided by e.g. Pascolo S. 2006; Stan R.2006, or A Mandavi 2006. Polynucleotide vaccines are easy to prepare,but the mode of action of these vectors in inducing an immune responseis not fully understood. Suitable vectors and delivery systems includeviral DNA and/or RNA, such as systems based on adenovirus, vacciniavirus, retroviruses, herpes virus, adeno-associated virus or hybridscontaining elements of more than one virus. Non-viral delivery systemsinclude cationic lipids and cationic polymers and are well known in theart of DNA delivery. Physical delivery, such as via a “gene-gun,” mayalso be used. The peptide or peptides encoded by the nucleic acid may bea fusion protein, for example with an epitope that stimulates T cellsfor the respective opposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CTLs and helper-T(T_(H)) cells to an antigen, and would thus be considered useful in themedicament of the present invention. Suitable adjuvants include, but arenot limited to 1018 ISS, aluminium salts, Amplivax, AS15, BCG,CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived fromflagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod (ALDARA), ImuFactIMP321, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system, PLGmicroparticles, resiquimod, SRL172, Virosomes and other Virus-likeparticles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21stimulon, which is derived from saponin, mycobacterial extracts andsynthetic bacterial cell wall mimics, and other proprietary adjuvantssuch as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's orGM-CSF are preferred. Several immunological adjuvants (e.g., MF59)specific for dendritic cells and their preparation have been describedpreviously (Dupuis M et al. 1998; Allison 1998). Also cytokines may beused. Several cytokines have been directly linked to influencingdendritic cell migration to lymphoid tissues (e.g., TNF-α), acceleratingthe maturation of dendritic cells into efficient antigen-presentingcells for T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No.5,849,589, specifically incorporated herein by reference in itsentirety) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23,IL-7, IFN-alpha, IFN-beta) (Gabrilovich et al. 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without bound bytheory, CpG oligonucleotides act by activating the innate (non-adaptive)immune system via Toll-like receptors (TLR), mainly TLR9. CpG triggeredTLR9 activation enhances antigen-specific humoral and cellular responsesto a wide variety of antigens, including peptide or protein antigens,live or killed viruses, dendritic cell vaccines, autologous cellularvaccines and polysaccharide conjugates in both prophylactic andtherapeutic vaccines. More importantly it enhances dendritic cellmaturation and differentiation, resulting in enhanced activation ofT_(H1) cells and strong cytotoxic T-lymphocyte (CTL) generation, even inthe absence of CD4 T cell help. The T_(H1) bias induced by TLR9stimulation is maintained even in the presence of vaccine adjuvants suchas alum or incomplete Freund's adjuvant (IFA) that normally promote aT_(H2) bias. CpG oligonucleotides show even greater adjuvant activitywhen formulated or co-administered with other adjuvants or informulations such as microparticles, nano particles, lipid emulsions orsimilar formulations, which are especially necessary for inducing astrong response when the antigen is relatively weak. They alsoaccelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg et al. 2006). U.S. Pat. No. 6,406,705 B1 describesthe combined use of CpG oligonucleotides, non-nucleic acid adjuvants andan antigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and AmpliGen, non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4 and SC58175, which mayact therapeutically and/or as an adjuvant. The amounts andconcentrations of adjuvants and additives useful in the context of thepresent invention can readily be determined by the skilled artisanwithout undue experimentation.

Preferred adjuvants are dSLIM, Interferon-alpha, -beta, CpG7909, IC31,ALDARA (Imiquimod), PeviTer, RNA, tadalafil, temozolomide, andJuvImmune.

The present invention provides a medicament that useful in treatingcancer, preferably neuronal cancer, and most preferably brain cancer.The cancer may be non-metastatic or metastatic, in particular it may beastrocytoma, pilocytic astrocytoma, dysembryoplastic neuroepithelialtumor, oligodendrogliomas, ependymoma, glioblastoma multiforme, mixedgliomas, oligoastrocytomas, medulloblastoma, retinoblastoma,neuroblastoma, germinoma, teratoma, gangliogliomas, gangliocytoma,central gangliocytoma, primitive neuroectodermal tumors (PNET, e.g.medulloblastoma, medulloepithelioma, neuroblastoma, retinoblastoma,ependymoblastoma), tumors of the pineal parenchyma (e.g. pineocytoma,pineoblastoma), ependymal cell tumors, choroid plexus tumors,neuroepithelial tumors of uncertain origin (e.g. gliomatosis cerebri,astroblastoma) or glioblastoma.

Since the peptides of the invention were isolated from glioblastoma, andin the case of SEQ ID NO:2 also from adenosquamous carcinoma, themedicament of the invention is preferably used to treat glioblastoma oradenosquamous carcinoma.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from glioblastoma or in the case of SEQ ID NO:2 alsoisolated from adenousquamous carcinoma, and since it was determined thatthese peptides are not present in normal tissues, these peptides can beused to diagnose the presence of these types of cancer.

The presence of the peptides of the present invention on tissue biopsiescan assist a pathologist in diagnosis of cancer. Detection of certainpeptides of the present invention by means of antibodies, massspectrometry or other methods known in the art can inform thepathologist whether the tissue is malignant, inflamed or generallydiseased. The presence of groups of peptides of the present inventioncan enable classification or subclassification of diseased tissues.

The detection of the peptides of the present invention on diseasedtissue specimen can enable the decision about the benefit of therapiesinvolving the immune system, especially if T lymphocytes are known orexpected to be involved in the mechanism of action. Loss of MHCexpression is a well described mechanism by which infected of malignantcells escape immunosurveillance. Thus, presence of the peptides of thepresent invention shows that this mechanism is not exploited by theanalyzed cells.

The peptides of the present invention may be used to analyze lymphocyteresponses against those peptides of the present invention, such as Tcell responses or antibody responses against the peptides of the presentinvention or the peptides of the present invention complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate markers in immunotherapy approaches aiming to inducelymphocyte responses by different means, e.g. vaccination of protein,nucleic acids, autologous materials, adoptive transfer of lymphocytes.In gene therapy settings, lymphocyte responses against the peptides ofthe present invention can be considered in the assessment of sideeffects. Monitoring of lymphocyte responses might also be a valuabletool for follow-up examinations of transplantation therapies, e.g. forthe detection of graft versus host and host versus graft diseases.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes, such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues. In addition, the peptidescan be used to verify a pathologist's diagnosis of a cancer based on abiopsied sample.

In yet another aspect thereof, the present invention relates to a kitcomprising (a) a container that contains a pharmaceutical composition asdescribed above, in solution or in lyophilized form; (b) optionally, asecond container containing a diluent or reconstituting solution for thelyophilized formulation; and (c) optionally, instructions for (i) use ofthe solution or (ii) reconstitution and/or use of the lyophilizedformulation. The kit may further comprise one or more of (iii) a buffer,(iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe. Thecontainer is preferably a bottle, a vial, a syringe or test tube; and itmay be a multi-use container. The pharmaceutical composition ispreferably lyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contains instructions on or associated with thecontainer that indicates directions for reconstitution and/or use. Forexample, the label may indicate that the lyophilized formulation is toreconstituted to peptide concentrations as described above. The labelmay further indicate that the formulation is useful or intended forsubcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500n). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have a distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, a anti-angiogenesis agent orinhibitor, a apoptosis-inducing agent or a chelator) or a pharmaceuticalcomposition thereof; The components of the kit may be pre-complexed oreach component may be in a separate distinct container prior toadministration to a patient. The components of the kit may be providedin one or more liquid solutions, preferably, an aqueous solution, morepreferably, a sterile aqueous solution. The components of the kit mayalso be provided as solids, which may be converted into liquids byaddition of suitable solvents, which are preferably provided in anotherdistinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The pharmaceutical formulation of the present invention is one that issuitable for administration of the peptides by any acceptable route suchas oral (enteral), nasal, ophthal, subcutaneous, intradermal,intramuscular, intravenous or transdermal. Preferably the administrationis s.c., and most preferably, i.d. Administration may be by infusionpump.

Definitions

As used herein and except as noted otherwise, all terms are defined asgiven below.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are typically 9 amino acids in length, but can be as short as 8amino acids in length, and as long as 14 amino acids in length.

The term “oligopeptide” is used herein to designate a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.The length of the oligopeptide is not critical to the invention as longas the correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 14 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to proteinmolecules of longer than about 30 residues in length.

A peptide, oligopeptide, protein, or polynucleotide coding for such amolecule is “immunogenic” (and thus an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a CTL-mediated response. Thus, an “immunogen”would be a molecule that is capable of inducing an immune response, andin the case of the present invention, a molecule capable of inducing aCTL response.

A T cell “epitope” is a short peptide molecule that binds to a class Ior II MHC molecule and that is subsequently recognized by a T cell. Tcell epitopes that bind to class I MHC molecules are typically 8-14amino acids in length, and most typically 9 amino acids in length. Tcell epitopes that bind to class II MHC molecules are typically 12-30amino acids in length. In the case of epitopes that bind to class II MHCmolecules, the same T cell epitope may share a common core segment, butdiffer in the length of the carboxy- and amino-terminal flankingsequences due to the fact that ends of the peptide molecule are notburied in the structure of the class II MHC molecule peptide-bindingcleft as they are in the class I MHC molecule peptide-binding cleft.

There are three different genetic loci that encode for MHC class 1molecules: HLA-A, HLA-B, and HLA-C. HLA-A1, HLA-A2, and HLA-A11 areexamples of different class I MHC molecules that can be expressed fromthese loci.

As used herein, reference to a DNA sequence includes both singlestranded and double stranded DNA. Thus, the specific sequence, unlessthe context indicates otherwise, refers to the single strand DNA of suchsequence, the duplex of such sequence with its complement (doublestranded DNA) and the complement of such sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be from a normal, mutated or altered gene, or caneven be from a DNA sequence, or gene, wholly synthesized in thelaboratory using methods well known to those of skill in the art of DNAsynthesis.

The term “nucleotide sequence” refers to a heteropolymer ofdeoxyribonucleotides.

The nucleotide sequence encoding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene which is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment,” when referring to a coding sequence, means aportion of nucleic acid comprising less than the complete coding regionwhose expression product retains essentially the same biologicalfunction or activity as the expression product of the complete codingregion.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnontranslated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that is pairedwith one strand of DNA and provides a free 3′OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, the claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly contemplated.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form.” As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform.

The term “active fragment” means a fragment that generates an immuneresponse (i.e., has immunogenic activity) when administered, alone oroptionally with a suitable adjuvant, to an animal, such as a mammal, forexample, a rabbit or a mouse, and also including a human, such immuneresponse taking the form of stimulating a CTL response within therecipient animal, such as a human. Alternatively, the “active fragment”may also be used to induce a CTL response in vitro.

As used herein, the terms “portion,” “segment,” and “fragment,” whenused in relation to polypeptides, refer to a continuous sequence ofresidues, such as amino acid residues, which sequence forms a subset ofa larger sequence. For example, if a polypeptide were subjected totreatment with any of the common endopeptidases, such as trypsin orchymotrypsin, the oligopeptides resulting from such treatment wouldrepresent portions, segments or fragments of the starting polypeptide.This means that any such fragment will necessarily contain as part ofits amino acid sequence a segment, fragment or portion, that issubstantially identical, if not exactly identical, to a sequence of SEQID NO: 1 to 11, which correspond to the naturally occurring, or “parent”proteins of the SEQ ID NO: 1 to 11. When used in relation topolynucleotides, such terms refer to the products produced by treatmentof the polynucleotides with any of the common endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical,” when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The Percent Identity isthen determined according to the following formula:

Percent Identity=100 [I−(C/R)] wherein C is the number of differencesbetween the Reference Sequence and the Compared Sequence over the lengthof alignment between the Reference Sequence and the Compared Sequencewherein (i) each base or amino acid in the Reference Sequence that doesnot have a corresponding aligned base or amino acid in the ComparedSequence and (ii) each gap in the Reference Sequence and (iii) eachaligned base or amino acid in the Reference Sequence that is differentfrom an aligned base or amino acid in the Compared Sequence, constitutesa difference; and R is the number of bases or amino acids in theReference Sequence over the length of the alignment with the ComparedSequence with any gap created in the Reference Sequence also counted asa base or amino acid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity, then theCompared Sequence has the specified minimum percent identity to theReference Sequence, even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified PercentIdentity.

The original peptides disclosed herein can be modified by thesubstitution of one or more residues at different, possibly selective,sites within the peptide chain. Such substitutions may be of aconservative nature, for example, where one amino acid is replaced by anamino acid of similar structure and characteristics, such as where ahydrophobic amino acid is replaced by another hydrophobic amino acid.Even more conservative would be replacement of amino acids of the sameor similar size and chemical nature, such as where leucine is replacedby isoleucine. In studies of sequence variations in families ofnaturally occurring homologous proteins, certain amino acidsubstitutions are more often tolerated than others, and these are oftenshow correlation with similarities in size, charge, polarity, andhydrophobicity between the original amino acid and its replacement, andsuch is the basis for defining “conservative substitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1—small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2—polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3—polar,positively charged residues (His, Arg, Lys); Group 4—large, aliphatic,nonpolar residues (Met, Leu, lie, Val, Cys); and Group 5—large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly nonconservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character.

Such radical substitutions cannot, however, be dismissed as potentiallyineffective since chemical effects are not totally predictable andradical substitutions might well give rise to serendipitous effects nototherwise predictable from simple chemical principles.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,amino acids possessing non-standard R groups (i.e., R groups other thanthose found in the common 20 amino acids of natural proteins) may alsobe used for substitution purposes to produce immunogens and immunogenicpolypeptides according to the present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan four positions within the peptide would simultaneously besubstituted.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted CTLs, effector functions may be lysisof peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation. For MHC class II-restricted T helper cells,effector functions may be peptide induced secretion of cytokines,preferably, IFN-gamma, TNF-alpha, IL-4, IL5, IL-10, or IL-2, orpeptide-induced degranulation. Possible effector functions for CTLs andT helper cells are not limited to this list.

Based on cytotoxicity assays, an epitope is considered substantiallyidentical to the reference peptide if it has at least 10% of theantigenic activity of the reference peptide as defined by the ability ofthe substituted peptide to reconstitute the epitope recognized by a CTLin comparison to the reference peptide. Thus, when comparing the lyticactivity in the linear portion of the effector:target curves withequimolar concentrations of the reference and substituted peptides, theobserved percent specific killing of the target cells incubated with thesubstituted peptide should be equal to that of the reference peptide atan effector:target ratio that is no greater than 10-fold above thereference peptide effector:target ratio at which the comparison is made.

Preferably, when the CTLs specific for a peptide of SEQ ID NO: 1 to 11are tested against the substituted peptides, the peptide concentrationat which the substituted peptides achieve half the maximal increase inlysis relative to background is no more than about 1 mM, preferably nomore than about 1 μM, more preferably no more than about 1 nM, and stillmore preferably no more than about 100 pM, and most preferably no morethan about 10 pM. It is also preferred that the substituted peptide berecognized by CTLs from more than one individual, at least two, and morepreferably three individuals.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

It should be understood that the features of the invention as disclosedand described herein can be used not only in the respective combinationas indicated but also in a singular fashion without departing from theintended scope of the present invention. For the purposes of the presentinvention, all references as cited herein are incorporated by referencein their entireties.

The following examples are provided for illustrative purposes only andare not intended to limit the invention.

EXAMPLES Example 1: Identification of Tumor Associated Peptides (TUMAPs)Presented on Cell Surface Tissue Samples

Patients' tumor and healthy tissues were provided by Hôpital CantonalUniversitaire de Genève (Medical Oncology Laboratory of TumorImmunology) and Neurochirurgische Universitats-Klinik Heidelberg(Molekularbiologisches Labor). Written informed consents of all patientshad been given before surgery. Tissues were shock-frozen in liquidnitrogen immediately after surgery and stored until isolation of TUMAPsat −80° C.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk, K. et al. 1991; Seeger, F. H. et al., 1999) using theHLA-A*02-specific antibody BB7.2 or the HLA-A, -B, -C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment and ultrafiltration.

Detection of TUMAPs by ESI-Liquid Chromatography Mass Spectrometry(ESI-LCMS) Method One:

The obtained HLA peptide pools were separated according to theirhydrophobicity by reversed-phase chromatography (CapLC, Waters) and theeluting peptides were analyzed in a hybrid quadrupole orthogonalacceleration time of flight tandem mass spectrometer (Q-TOF Ultima,Waters) equipped with an ESI source. Peptide pools were loaded onto aC18 pre-column for concentration and desalting. After loading, thepre-column was placed in line for separation by a fused-silicamicro-capillary column (75 μm i.d.×250 mm) packed with 5 μm C18reversed-phase material (Dionex). Solvent A was 4 mM ammoniumacetate/water. Solvent B was 2 mM ammonium acetate in 80%acetonitrile/water. Both solvents were adjusted to pH 3.0 with formicacid. A binary gradient of 15% to 60% B within 90 minutes was performed,applying a flow rate of 5 μl/min reduced to approximately 200 nl/min bya split-system. A gold coated glass capillary (PicoTip, New Objective)was used for introduction into the micro-ESI source. The integrationtime for the TOF analyzer was 1.9 s with an interscan delay of 0.1 s.Subsequently, the peptide sequences were revealed by collisionallyinduced decay (CID) mass spectrometry (ESI-LCMS/MS). The identifiedTUMAP sequence was assured by comparison of the generated natural TUMAPfragmentation pattern with the fragmentation pattern of a syntheticsequence-identical reference peptide.

Method Two:

The obtained HLA peptide pools were separated according to theirhydrophobicity by reversed-phase chromatography (Acquity UPLC system,Waters) and the eluting peptides were analyzed in LTQ-Orbitrap hybridmass spectrometer (ThermoElectron) equipped with an ESI source. Peptidepools were loaded directly onto the analytical fused-silicamicro-capillary column (75 μm i.d.×250 mm) packed with 1.7 μm C18reversed-phase material (Waters) applying a flow rate of 400 nL perminute. Subsequently, the peptides were separated using an two-step 180minute-binary gradient from 10% to 33% B at flow rates of 300 nL perminute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe micro-ESI source. The LTQ-Orbitrap mass spectrometer was operated inthe data-dependent mode using a TOPS strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30,000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified TUMAP sequence wasassured by comparison of the generated natural TUMAP fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

FIGS. 1a and 1b show exemplary spectra obtained from tumor tissue forMHC class I associated TUMAPs.

TABLE 3 List of tumor samples on which the peptides were identified SEQID NO Peptide Code Tumor Sources 1 PTP-001 GB1006T, GB1012T 2 PTP-002GB1023T, GB6003T 3 PTP-003 GB1008T, GB1011T, GB1012T, GB1014T, GB1020T,GB1021T, GB1023T, GB1026T, GB6003T, GB6010T, GB6015T, GB6016T, GB6019T,GB6024T, GB6027T 4 PTP-004 GB1023T, GB6010T 5 PTP-005 GB1023T, GB6010T,GB6027T 6 PTP-006 GB1012T 7 PTP-007 GB1023T, GB6010T, GB6027T 8 PTP-008NCH361T 9 PTP-009 GB6003T 10 PTP-010 GB6010T 11 CHI-001 GB1002, GB1020T,GB1021T, GB1023T, GB1026T, GB6003T, GB6010T, GB6027T,

Example 2: Expression Profiling of Genes Encoding the Peptides of theInvention

Not all peptides identified as presented on the surface of tumor cellsby MHC molecules are suitable for immunotherapy, because the majority ofthese peptides are derived from normal cellular proteins expressed bymany cell types. Only few of these peptides are tumor-associated andlikely able to induce T cells with a high specificity of recognition forthe tumor from which they were derived. To identify such peptides andminimize the risk for autoimmunity induced by vaccination, the inventorsfocused on those peptides that are derived from proteins that areover-expressed on tumor cells compared to the majority of normaltissues.

The ideal peptide is derived from a protein that is unique to the tumorand not present in any other tissue. To identify peptides that arederived from genes with an expression profile similar to the ideal, theidentified peptides were assigned to the proteins and genes,respectively, from which they were derived and expression profiles ofthese genes were generated.

RNA Sources and Preparation

Surgically removed tissue specimens were provided by two differentclinical sites (see Example 1) after written informed consent had beenobtained from each patient.

Tumor tissue specimens were snap-frozen in liquid nitrogen immediatelyafter surgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRIzol(Invitrogen, Karlsruhe, Germany) followed by a cleanup with RNeasy(QIAGEN, Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed so that RNA fromeach individual was equally weighted. Leukocytes were isolated fromblood samples of 4 healthy volunteers.

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

Microarray Experiments

Gene expression analysis of all tumor and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual. Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip NT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analysed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalisation, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal sample was arbitrarily set to 1.0.

The expression profile of the source gene of all peptides of the presentinvention (PTPRZ 1) shows a high expression in glioblastoma tumor tissuewhereas the gene is not expressed or expressed at very low levels innormal tissues FIG. 2).

Example 3: In Vitro Immunogenicity of MHC Class I Presented Peptides

In Vitro Priming of CD8+ T Cells

To perform in vitro stimulations by artificial antigen presenting cells(aAPC) loaded with peptide-MHC complex (pMHC) and anti-CD28 antibody,PBMCs (peripheral blood mononuclear cells) were isolated from freshHLA-A*02+ buffy coats by using standard density gradient separationmedium (PAA, Cölbe, Germany). Buffy coats were obtained from theKatharinenhospital Stuttgart. Isolated PBMCs were incubated overnight inT-cell medium (TCM) for human in vitro priming consisting ofRPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10%heat inactivated human AB serum (PAA, Cölbe, Germany), 100 U/mlPenicillin/100 μg/ml Streptomycin (Cambrex, Verviers, Belgium), 1 mMsodium pyruvate (CC Pro, Neustadt, Germany) and 20 μg/ml Gentamycin(Cambrex). CD8+ lymphocytes were isolated using the CD8+ MACS positiveselection kit (Miltenyi, Bergisch Gladbach, Germany) according to themanufacturer's instructions. Obtained CD8+ T cells were incubated untiluse in TCM supplemented with 2.5 ng/ml IL-7 (PromoCell, Heidelberg,Germany) and 10 U/ml IL-2 (Chiron, Munich, Germany). Generation ofpMHC/anti-CD28 coated beads, T-cell stimulations and readout wasperformed as described before (Walter et. al., 2003) with minormodifications. Briefly, biotinylated recombinant HLA-A*0201 moleculeslacking the transmembrane domain and biotinylated at the carboxyterminus of the heavy chain were produced following a method describedby Altman et al., 1996). The purified costimulatory mouse IgG2a antihuman CD28 Ab 9.3 (Jung et al., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). 5.60 μm large streptavidin coated polystyreneparticles beads were used (Bangs Laboratories, Illinois/USA). pMHC wasused as positive and negative controls were A*0201/MLA-001 (peptideELAGIGILTV from modified Melan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHIfrom DDX5), respectively.

800,000 beads/200 μl were coated in 96-well plates in the presence of600 ng biotin anti-CD28 plus 200 ng relevant biotin-pMHC (high densitybeads) or 2 ng relevant plus 200 ng irrelevant (pMHC library) MHC (lowdensity beads). Stimulations were initiated in 96-well plates byconincubating 1×10⁶ CD8+ T cells with 2×10⁵ washed coated beads in 200μl TCM supplemented with 5 ng/ml IL-12 (PromoCell) for 3-4 days at 37°C. Half of the medium was then exchanged by fresh TCM supplemented with80 U/ml IL-2 and incubating was continued for 3-4 days at 37° C. Thisstimulation cycle was performed for a total of three times. Finally,tetrameric analyses were performed with fluorescent MHC tetramers(produced as described by (Altman et al., 1996)) plus antibody CD8-FITCclone SK1 (BD, Heidelberg, Germany) on a FACSCalibur or a LSR II flowcytometer (BD). Peptide specific cells were calculated as percentage oftotal CD8+ T cells. Evaluation of tetrameric analysis was performedusing the software FCS Express (De Novo Software). In vitro priming ofspecific tetramer+ CD8+ lymphocytes was detected by appropriate gatingand by comparing to negative control stimulations. Immunogenicity for agiven antigen was detected if at least one evaluable in vitro stimulatedwell of one healthy donor was found to contain a specific CD8+ T-cellline after in vitro stimulation (i.e. this well contained at least 1% ofspecific tetramer+ among CD8+ T cells and the percentage of specifictetramer+ cells was at least 10× the median of the negative controlstimulations).

A representative staining showing generation of T-cell lines specificfor PTP-002 and PTP-001 is shown in FIGS. 3a-3d . The results aresummarized in table 4 below.

TABLE 4 In vitro immunogenicity of peptides of the inventionImmunogenicity Positive donors/ Positive wells/wells Antigen detecteddonors tested tested PTP-001 Yes 6/6 (100%) 33/96 (34%) PTP-002 Yes 3/4(75%)   9/48 (19%) PTP-003 Yes 2/4 (50%)   8/48 (17%) PTP-004 Yes 2/4(50%)  2/48 (4%) PTP-005 Yes 4/4 (100%) 25/48 (52%) CHI-001 Yes 4/4(100%) 39/62 (63%)

Results of in vitro immunogenicity experiments conducted by immatics andshowing the percentage of positive tested donors and wells aresummarized here. Results shown have been obtained by stimulation of CD8+cells with high density beads. As different human serum lots may highlyaffect the immunogenicity results, only assays in which one and the sameserum lot was used, were evaluated together.

Example 4: Binding of HLA Class I-Restricted Peptides to HLA-A*0201

The objective of this analysis was to evaluate the affinity of HLA classI peptides PTP-001, PTP-002, PTP-003, PTP-004, PTP-005 and CHI-001 tothe MHC molecule coded by the HLA-A*0201 allele. Affinities for allpeptides to HLA-A*0201 were comparable to the well-known control peptideHBV-001, dissociations constants (K_(D)) are in the range from 0.02 to1.6 nM.

Principle of Test

Stable HLA/peptide complexes consist of three molecules: HLA heavychain, beta-2 microglobulin (b2m) and the peptidic ligand. The activityof denatured recombinant HLA-A*0201 heavy chain molecules alone can bepreserved making them functional equivalents of “empty HLA-A*0201molecules.” When diluted into aqueous buffer containing b2m and anappropriate peptide, these molecules fold rapidly and efficiently in anentirely peptide-dependent manner. The availability of these moleculesis used in an ELISA-based assay to measure the affinity of interactionbetween peptide and HLA class I molecule (Sylvester-Hvid et al., 2002).

Purified recombinant HLA-A*0201 molecules were incubated together withb2m and graded doses of the peptide of interest. The amount of denovo-folded HLA/peptide complexes was determined by a quantitativeELISA. Dissociation constants (K_(D) values) were calculated using astandard curve recorded from dilutions of a calibrant HLA/peptidecomplex.

Results are shown in FIG. 4. A lower K_(D) value reflects higheraffinity to HLA-A*0201. Affinities for all peptides to HLA-A*0201 werecomparable to the well-known control peptide HBV-001, dissociationsconstants (K_(D)) are in the range from 0.02 to 1.6 nM.

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1. A method of treating a patient who has glioblastoma, comprisingadministering to said patient a population of activated CD8+ cytotoxic Tcells that kill the cancer cells that aberrantly express a peptideconsisting of the amino acid sequence of AIIDGVESV (SEQ ID NO: 3) on thecell surface, wherein the peptide is in a complex with an MHC class Imolecule.
 2. The method of claim 1, wherein the activated CD8+ cytotoxicT cells are autologous to the patient.
 3. The method of claim 1, whereinthe activated CD8+ cytotoxic T cells are obtained from a healthy donor.4. The method of claim 1, wherein the activated CD8+ cytotoxic T cellsare derived from tumor infiltrating lymphocytes or peripheral bloodmononuclear cells.
 5. The method of claim 1, wherein the activated CD8+cytotoxic T cells are expanded in vitro.
 6. The method of claim 1,wherein the population of activated T cells are administered in the formof a composition.
 7. The method of claim 6, wherein the compositionfurther comprises an adjuvant.
 8. The method of claim 7, wherein theadjuvant is selected from anti-CD40 antibody, imiquimod, resiquimod,GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha,interferon-beta, CpG oligonucleotides and derivates, poly-(I:C) andderivates, RNA, sildenafil, particulate formulations with poly(lactideco-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-5,IL-7, IL-10, IL-12, IL-15, and IL-23.
 9. The method of claim 1, whereinthe activated CD8+ cytotoxic T cells are produced by contacting T cellswith an antigen presenting cell that expresses the peptide in a complexwith an MHC class I molecule on the surface of the antigen presentingcell, for a period of time sufficient to activate said T cell.
 10. Themethod of claim 9, wherein the antigen presenting cell is infected witha recombinant virus expressing the peptide.
 11. The method of claim 10,wherein the antigen presenting cell is a dendritic cell or a macrophage.12. The method of claim 5, wherein the expansion is in the presence ofan anti-CD28 antibody and IL-12.
 13. The method of claim 9, wherein thecontacting is in vitro.
 14. The method of claim 1, wherein the MHC classI molecule is HLA-A*02.
 15. The method of claim 8, wherein the adjuvantcomprises IL-7.
 16. The method of claim 8, wherein the adjuvantcomprises IL-12.
 17. A method of treating a patient who glioblastoma,comprising administering to said patient a composition comprising apeptide in the form of a pharmaceutically acceptable salt and anadjuvant, wherein said peptide consists of the amino acid sequence ofAIIDGVESV (SEQ ID NO: 3), thereby inducing a T-cell response to theglioblastoma.
 18. The method of claim 17, wherein the T cell response isa cytotoxic T cell response.
 19. The method of claim 15, wherein theadjuvant is selected from anti-CD40 antibody, imiquimod, resiquimod,GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha,interferon-beta, CpG oligonucleotides and derivates, poly-(I:C) andderivates, RNA, sildenafil, particulate formulations with poly(lactideco-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-5,IL-7, IL-10, IL-12, IL-15, and IL-23.
 20. The method of claim 19,wherein the adjuvant comprises IL-7.